Agents for treating osteoporosis and inhibiting osteoclast formation

ABSTRACT

Compositions and methods for treating osteoporosis, the formation of osteoeclasts, and/or for the suppression of decreasing spongy bone density. Example methods include administering  Cordyceps sinensis  or its processed product.

CLAIM FOR PRIORITY

This application is a continuation of U.S. Application Ser. No.10/825,585 filed on Apr. 16, 2004, which claims priority to Japanesepatent application 2003-112655 filed on Apr. 17, 2003, both of which areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention is related to Cordyceps Sinensis (C. Sinensis)that can prevent the manifestation of osteoporosis. In particular, thepresent invention is related to the composition for treatingosteoporosis and osteoclast formation comprising C. Sinensis mycelium asthe effective ingredient.

BACKGROUND OF THE INVENTION

The function of bone is to support body against gravity and to conductmovement. As the largest reservoir of calcium in the body, the bone alsomaintains the balance of calcium in the body. Because of having movementfunction and maintaining the balance of calcium in the body, the renewaland resorption of bone and the bone will be made repeatedly. Duringchildhood and the adolescent, bone formation is more important than boneresorption, thus bone grows larger, heavier and denser.

The following briefly states how the bone goes through its life cyclethrough different stages in bone remodeling.

The bone remodeling is mainly regulated by chondrocyte, osteoblast andosteoclast. The bone remodeling can be divided into three stages. Thatis, (1) bone renewal and growth due to intrachondral bone formation, (2)the cycle of remodeling bone formation and bone resorption to maintainquality and quantity; and (3) re-growth during bone injury such asfracture.

Each stages described above is regulated by various Ca²⁺-dependentenzymes, cytokines, gene transcription and other cellular regulationmechanisms to regulate activities the differentiation of the bone.

For example, the remodeling cycle involves bone resorption performed byosteoclasts. Osteoclasts remove the old or worn-out mineralized bone.Bone resorption is initiated when osteoclasts receive signals from thesurface of bone that are stimulated by enzymes or cytokines. On theother hand, osteoblasts rebuild new bone tissue by laying down anunmineralized matrix, called osteoid, which will eventually form newmineralized bone.

When this rebuilding is complete, the area of bone remodeling restsuntil the next remodeling cycle begins. Under normal condition, boneresorption and formation are performed at the same time, thus the bonecan renew without affecting bone's function.

As we march into the elder-populated society, osteoporosis that happensat youth is drawing more and more attention. Osteoporosis is not adisease of change in the bone component, but a decrease of bone mass.The decrease of bone density and bone strength make one more susceptibleto accident and the resulting fracture or bone damages. The primarycause of osteoporosis is the imbalance of bone remodeling, the overlyincrease of osteoclasts and bone resorption. In addition, many femalepatients at menopause suffered from osteoporosis, suggesting that thelower of sex hormone (i.e. estrogen) is one of the causes ofosteoporosis.

There are currently many agents available for osteoporosis on themarket. For example, estrogen can treat osteoporosis. Therefore, steroidenzymes and derivatives similar to chemical structure of estrogen areregarded as potential agents for treating osteoporosis. In theory, theenzymes are not only applied to treat osteoporosis, but decrease theside effects caused by estrogen. However, the substantive examinationhas not studied yet.

In addition, JP H04-352795 disclosed agents that are hydroxyl containingsteroidal hormones, having bone resorption antagonist or bone formationstimulatory activity. The agent acts as bone resorption antagonist.However, the actual experimental data have not been disclosed. JPH07-215878 disclosed an agent containing mepitiostane and epitiostanolas active ingredients for treating osteoporosis.

JP H08-12580 is related to a synostosis promotor containing a specificactive vitamin D and its derivative as active ingredients, promotingbone-repairing step after extension of bone, cut of bone, fracture,etc., and useful for shortage of treating period and prevention andtreatment for re-fracture.

JP H11-60489 disclosed a medicine for preventing/treating osteoporosiswhich enhances density and strength of bone without elevating theconcentration of calcium in blood, by including an active type vitamin Dand bisphosphonic acid as active ingredients.

However, the prior art above provides chemical compounds only, thereforecannot sufficiently treat osteoporosis.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides a composition for treating osteoporosiscomprising C. Sinensis or the processed product of C. Sinensis as theeffective ingredient.

The present invention further provides a composition for suppressing theformation of osteoclasts comprising C. Sinensis or the processed productof C. Sinensis as the effective ingredient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 discloses the procedure of preparing water extracts of themycelium of C. Sinensis.

FIG. 2 discloses bone density of lumbar of rats from Sham (shamoperation) group, OVX (ovary-removed control) group and ovary-removedrats treated with water extracts of the mycelium of C. Sinensis 10, 50,100 mg/kg.

DETAILED DESCRIPTION OF THE INVENTION

After extensive researches, the inventor has found C. sinensis to beeffective in preventing osteoporosis. C. Sinensis, a plant of the ergotfamily, is a traditional and precious dried Chinese medicinal herbbelonging to the fungus category. There are more than 300 species in theworld, and the host in which C. Sinensis resides varies from insects tofungus. Depending on different hosts, the type of C. Sinensis parasitescan be different. Sometimes different species of C. Sinensis resides onspecifically the lava or the adult of the same insect.

As described above, many kinds of C. Sinensis are known to supplementhealth, provide energy and cure anemia. Recent researches have alsoshown that C. Sinensis has anti-tuberculosis bacterium activity and canexpand bronchi as well as increase adrenaline secretion whenadministered.

The inventor has discovered another therapeutic effect of C. Sinensis.By administering certain amount of agent comprising the mycelium of C.Sinensis cultured and fermented by the specific process of theinvention, the formation of osteoclasts was suppressed. In addition, thedecrease of osteoclast formation is expected to rebalance boneresorption and bone formation, thereby preventing the decrease of bonedensity and improving osteoporosis.

Therefore, the invention provides a composition for treatingosteoporosis comprising C. Sinensis or the processed product of C.Sinensis as the effective ingredient. The treatment of osteoporosis ismade by suppressing the decrease of spongy bone density.

The invention further provides a composition for preventing theformation of osteoclasts comprising C. Sinensis or the processed productof C. Sinensis as the effective ingredient.

The invention further provides a composition for suppressing thedecrease of spongy bone density comprising Cordyceps Sinensis or theprocessed product of Cordyceps Sinensis as the effective ingredient.

The preferred embodiment of C. Sinensis is the mycelium of C. Sinensis.The preferred embodiment of the processed product of C. Sinensis isprepared from water or organic solvent extract of mycelium of C.Sinensis. The organic solvent includes but is not limited to alcohol(such as CH₃OH, C₂H₅OH, C₃H₇OH), ester (such as acetyl acetate), alkane(such as hexane) and halogenated alkane (such as CH₃C₁, C₂H₂Cl₂). Thepreferred organic solvent is methanol, ethanol or alcoholic solventwithout causing any side effect of human.

The invention further provides the method of preparing the processedproduct of the mycelium of C. Sinensis. The method comprises culturingthe mycelium of C. Sinensis on slant culture or plate culture system.The preferred embodiment of the culture is the slant culture. The methodfurther comprises procedures of

-   1. Potato cubes, glucose and agar were added into distilled water to    prepare Potato Dextrose Agar (PDA)-   2. Culture

a. Slant culture

-   -   PDA was dissolved in hot water until completely dissolved. 8 ml        culture medium was added into 16×160 mm test tube. Then, the        mixture was sterilized. Germ was cut with sterilized blazer and        inoculated into slant medium. The medium was cultivated at 28°        C.

b. Plate culture

-   -   PDA was added in water and divided into 5 sterilized culture        flasks. After about 20 ml the culture medium was added to the        plate. Germ was cut out with sterilized blazer and inoculated        into slant medium. The medium was cultivated at 28° C.    -   The mycelium of C. Sinensis from the medium was processed at        cold temperature. The mycelium was separated from the medium.        The separated mycelium was dried and smashed to obtain the        powder of the mycelium of C. Sinensis.        The invention further provides the method of mass fermenting the        processed product of the mycelium of C. Sinensis. The method        comprises

-   1. Colonies were selected and grown in flasks.

-   2. After fermented in flask 1 and flask 2, large-scale fermentation    was performed in production tank. The conditions of the production    task were as follows: 96˜136 hours, pH 5.5˜6.0, air flow 0.2˜0.3    vvm, 25˜28° C.

-   3. The two stage mass production was fermented at 150˜180 rpm,    25˜28° C., for 8˜10 days at pH 5.5˜6.0.

-   4. The mycelium was separated from the culture medium. The dried    mycelium was smashed, filled, sealed, packed and stored.

The most preferred embodiment of the processed product of C. Sinensis isthe water extraction of C. Sinensis. The preferred concentration of thewater extract is above 30 μg/ml. It has been found that the suppressionof osteoclasts is less effective if the concentration is lower than 30μg/ml. The better concentration is 30-3000 μg/ml. The most preferredconcentration of the water extraction of the mycelium of C. Sinensis is300˜3000 μg/ml.

In addition, the composition of the invention further comprisesnon-toxic non active carriers in adjusted proportion to be administeredto animals including human. The carriers can be in solid, semi-solid orliquid phase. The carrier can be selected from more than one dilute,filler or adjuvant.

The invention can be administered via intravenous, oral, intra-tissue,eye, nose, or colon route. The preferred embodiment is administrationvia oral route.

Oral administration can be administered in solid or liquid form,including in the form of powder, tablet, capsule, granule, droplet,solution, syrup, sublingual tablets or in other forms. The preferredembodiment is in the form of powder.

The preferred amount of the composition of the invention administereddepends on the patient's condition and the administration route.

The preferred amount of the composition of the invention administratedto a normal adult is 25˜250 mg/kg per day. The more preferred amount is125˜250 mg/kg per day. The administration may be divided into twice orfour times a day.

EXAMPLES

First, the process of preparing the composition of the invention isdescribed. The following examples demonstrate that the mycelium of C.Sinensis suppresses the formation of osteoclasts in vitro. The nextexample demonstrates the change of bone density (measured by DEXA (Dualenergy X-ray absorptiometry) and pQCT (peripheral quantitative computedtomography)) after treating with the composition of the invention.

Example 1 The preparation of the Mycelium of C. Sinensis

The mycelium of C. Sinensis in this example could be cultured on slantculture or plate culture system.

-   1. 300.0 g potato cubes, 20.0 g glucose and 15.0 g agar were added    into 1.0 L distilled water to prepare Potato Dextrose Agar (PDA)-   2. Slant culture    -   39 g PDA was dissolved in hot water (1.0 L) until completely        dissolved. 8 ml culture medium was added into 16×160 mm test        tube. Then, the mixture was sterilized at 121° C. for 20 min.        0.5² mm of germ was cut with sterilized blazer and inoculated        into slant medium. The medium was cultivated at 28° C.-   3. Plate culture    -   39 g PDA was added in 1 L water and separated into 5 culture        flasks. The flasks were sterilized at 121° C. for 20 min. After        about 20 ml the culture medium was added to the plate. 0.5² mm        of germ was cut out with sterilized blazer and inoculated into        slant medium. The medium was cultivated at 28° C.    -   The mycelium of C. Sinensis from the medium was processed at        cold temperature. The mycelium was separated from the medium.        The separated mycelium was dried and smashed to obtain the        powder of the mycelium of C. Sinensis.-   4. Mass fermentation    -   Colonies were selected and were grown in flasks. After fermented        in flask 1 and flask 2, large-scale fermentation was performed        in production tank. The mycelium was separated from the culture        medium. The dried mycelium was smashed, filled, sealed, packed        and stored. The conditions of the production task were as        follows: 96˜136 hours, pH 5.5˜6.0, air flow 0.2˜0.3 vvm,        25˜28° C. The two stage mass production was fermented at 150˜180        rpm, 25˜28° C., for 8˜10 days at pH 5.5˜6.0. In addition, water,        starch, glucose and extractions of animal or plant proteins        could be used in the culture medium.

Example 2 Water Extraction of C. sinensis Could Suppress the Developmentof Osteoclasts

Myelocytes and osteoblasts were co-cultured in vitro and the numbers ofosteoclasts formed were measured. Myelocytes will differentiate intoosteoclasts expressing tartrate-resisted acidic phosphatase (TRAP) whenvitamin D is present in the coculture. These osteoclasts could beidentified by staining for TRAP. Therefore the development ofosteoclasts could be assayed by co-culturing myelocytes withosteoblasts.

Example 3 Preparation of Water Extraction of the Mycelium of C. sinensis

FIG. 1 showed the procedure of preparing the water extraction. First, 80g of the mycelium of C. Sinensis was extracted by 1200 ml of diethylether until reflux. The extraction was repeated two times. Next, residuewas extracted by 1200 ml of methanol until reflux. Likewise, themethanol extraction was formulated. Residue was extracted by 1200 ml ofdistilled water until reflux. Likewise, the water extraction wasformulated.

Example 4 Co-Culture Procedure

Osteoblasts and, myelocytes were co-cultured in 24-well plate. PTH (100ng/ml), 1α, 25 (OH)₂VD₃ (10⁻⁸ M), water extraction of the mycelium of C.Sinensis 3 mg/ml, 300 μg/ml, 30 μg/ml, 3 μg/ml or Elcatonin 2 U/ml wereadded. Samples were numbered No. 1˜No. 6. Each sample was cultured in 4wells. Culture was maintained for 6 days with medium changed every twodays.

Example 5 Identification of Osteoclasts Formation

After coculturing, TRAP-positive cells were stained to dark red color.The numbers of TRAP-positive osteoclasts that have more than 3 nucleuseswere counted. The average numbers of osteoclasts from 4 samples were thenumber of osteoclasts.

The numbers of osteoclasts in the 1α, 25 (OH)₂D₃ sample was considered100%. The percentage of osteoclasts formed of experimental group wascalculated using formula 1. The result was shown in Table 1. Student'st-test was used to examine the significance.

The formation of osteoclasts (%) osteoclast cell numbers/osteoclast cellnumbers in medium with 1α,25(OH)₂D₃×100 (formula 1)

TABLE 1 Average Osteoclast cell Formation of Group No. Treatment numbers(n = 4) osteoclasts (%) T-test SD^(#) Exp. 1 The mycelium of C. Sinensis0.0 0.0 0.00 0.0 3 mg/ml 2 The mycelium of C. Sinensis 509.0 62.1 0.0030.9 300 μg/ml 3 The mycelium of C. Sinensis 478.5 58.4 0.00 108.9 30μg/ml 4 The mycelium of C. Sinensis 792.8 96.8 0.59 37.3 3 μg/ml Control5 1α,25(OH)₂D₃ 10⁻⁸ M 819.0 100.0 69.4 6 Elcatonin 2 U/ml 113.0 13.80.00 15.7 ^(#)SD: Standard deviation.

As shown in Table 1, the formation of osteoclasts in the positivecontrol—medium added with Elcatonin—was significantly different from thenegative control (1α, 25(OH)₂D₃). The formation of osteoclasts in mediumadded with 30 μg/ml, 300 μg/ml and 3 mg/ml water extraction of C.Sinensis mycelium were 58.4% 62.1% and 0.0%, respectively. Thus, it isclear that water extractions of the mycelium of C. Sinensis cansignificantly suppress the formation of osteoclasts. The effect ofadding 3 mg/ml water extraction of the mycelium of C. Sinensis (00) waseven better than Elcatonin (13.8%). According to the in vitro tests, itsuggest that water extraction of the mycelium of C. Sinensis myceliumsuppressed the formation of osteoclasts against osteoporosis.

Example 6 Use Dual Energy X-Ray Absorptiometry (DEXA) to Measure theEffect of the Water Extraction of the Mycelium of C. Sinensis In Vivo

Rats with ovaries removed were used as the model of osteoporosis. Therewere three groups to be tested. One control group was rats with shamoperations, two other groups were rats with ovaries removed. One of theovaries-removed groups was treated with water extraction of C. Sinensis.Whether the treatment can effectively lower one of the symptoms ofosteoporosis—to decrease bone density, was evaluated by measuring thetotal bone density of lumbar. The total bone density was measured by thestandard clinical technique DEXA.

The experiment procedures were as follows:

-   1. Eight month old rats were divided into 3 groups: ovary-removed    (OVX) group, Sham group and ovary-removed group treated with water    extractions of the mycelium of C. Sinensis. Each group included 7    rats.-   2. First rats were anesthetized by intraperitonealy injecting    amobarbital. Ovaries of rats in the OVX group were removed. Rats in    the Sham group were also anesthetized, but their ovaries were not    removed, only touched by tweezers.-   3. 10 mg/kg, 50 mg/kg and 100 mg/kg water extractions of the    mycelium of C. Sinensis were orally administered to rats.    Administration was performed every 2 days for 5 weeks.-   4. After 5 weeks, rats were anesthetized again using amobarbital.    BMD (bone mass density) was measured by DEXA.

Anesthetized rats were put on sterilized table. DEXA was turned to smallanimal program (line specing and point resolution: 0.5×0.5 mm, scanspeed: 15 mm/sec) to scan lumber spines. The average bone density oflumbar spine 2 to 4 (L2-L4) was measured. The significance was evaluatedby Student's t-test.

-   5. Rats were put to euthanasia and ovaries were removed to measure    ovary weight.

The results are shown in FIG. 2 and Table 2.

TABLE 2 Water extraction of C. Sinensis mycelium (mg/kg) Sham OVX 100 5010 Initial weight 296 ± 4  298 ± 5 298 ± 3 298 ± 5  293 ± 6 Final weight302 ± 4  334 ± 6 340 ± 7 331 ± 10 329 ± 6 Ovary weight 306 ± 53  46 ± 3 51 ± 3 49 ± 3  48 ± 3 (mg)/100 gb.w. ^(#)p < 0.01 (compared to Sham)

As shown in FIG. 2 and Table 2, the body weights of rats in the OVX andSham group were significantly different as expected, so were their ovaryweights.

As shown in FIG. 2, there was a significant difference between OVX groupand the group treated with the mycelium of C. Sinensis. Although theslowing of the decrease of BMD was not correlated with the concentrationof the water extracts, the body and ovary weight was not affected. Theinhibition of decreasing bone density of lumbar spine was lacking. Giventhe above, in vivo test about anti-osteoporosis activity was studied.

Example 7 Compare Bone Density by Peripheral Quantitative ComputedTomography (pQCT)

Similar to the DEXA experiment described above, rats with ovariesremoved were administered with different concentrations of testsubstance.

In order to assess the manifestation of osteoporosis, the rat tibia wascategorized into cortical bone and spongy bone. The density of corticalbone and spongy bone were measured and compared. Other SSI indexes ofbone dymatics such as the length and bone strength of periosteum andendosteum were also compared. Bone density was measured by performingpQCT. The experiment procedure is described below.

-   -   1. 6-month-old female Wistar rats were grown for a week to        familiarize to the environment.    -   2. After one week, rats were divided into ovaries-removed group,        Sham group and ovaries-removed group. Each group included 7        rats.    -   3. One week after the operation, suspension of the mycelium        of C. Sinensis 100 mg/kg was orally administrated, 3 times a        week.    -   4. One month after the surgery, rats were anesthetized with 50        mg/kg amobarbital and bone dynamic data was measured by PQCT.    -   5. After completing the measure, rats were weighted and put to        euthanasia using amobarbital and blood was collected. Serum was        collected by centrifugation and was stored at −80° C.    -   6. Ovaries were removed and weighted.    -   7. Data was analyzed and statistically processed.

The result was shown in Table 3˜Table 8. Table 3 showed the body andovary weight. Table 4 showed the total bone mass, total bone density andsection area. Table 5 was the total bone mass and density of spongybone. Table 6 was the length of periosteum and endosteum. Table 7 wasthe bone mass, bone density and thickness of cortical bone of tibia.Table 8 showed the index of bone strength.

TABLE 3 Group Body weight (g) Ovary weight (g) Control 326.8 ± 17.5 0.16± 0.03 Sham 307.5 ± 25.0 0.48 ± 0.06 T-100 305.0 ± 12.6 0.15 ± 0.03T-100 is suspension of the mycelium of C. Sinensis 100 mg/kg (1% CMC).

TABLE 4 Group n Total bone mass Total bone density Tangential areaControl 8  8.63 ± 0.60^(#)  672.65 ± 13.81^(#) 13.70 ± 1.22 Sham 4 9.93± 1.06 753.22 ± 32.26 13.16 ± 0.91 T-100 7 8.36 ± 0.61 681.04 ± 47.0812.30 ± 1.21 ^(#)p < 0.01 (Compared to Sham)

As shown in Table 3, the body and uterus weights of mice in theovary-removed group (control) and the Sham groups were significantlydifferent, thus the ovary-removed mice were appropriate model organismsfor osteoporosis. In addition, as shown in Table 4, the total bone massand total bone density of tibia was not significantly different betweenmice of control group and the T-100 group. The total bone density,however, seemed to have increased.

TABLE 5 Group Spongy bone mass (mg/mm) Spongy bone density (mg/cm³)Control 0.85 ± 0.16 170.5 ± 27.8^(##) Sham 0.84 ± 0.15 244.5 ± 40.0 T-100 0.98 ± 0.21 210.3 ± 23.6*  ^(##)p < 0.01 (Compared to Sham) *p <0.05 (Compared to control)

As shown in Table 5, the major symptom of osteoporosis—the density ofspongy bone, was significantly lowered in the group administered withmycelium of C. Sinensis. Therefore the powder of C. Sinensis mycelium isable to inhibit osteoporosis.

TABLE 6 Group periosteum length endosteum length Control 12.53 ± 0.519.28 ± 0.54 Sham 12.85 ± 0.45 9.17 ± 0.24 T-100 12.42 ± 0.63 9.25 ± 0.65

TABLE 7 Group Cortical bone mass Cortical bone density cortical boneControl  6.62 ± 0.37^(#) 1173.87 ± 18.27  0.52 ± 0.02^(#) Sham 7.50 ±0.67 1161.16 ± 18.20 0.59 ± 0.04 T-100 6.27 ± 0.40 1146.62 ± 23.90 0.50± 0.01 ^(#)p < 0.05 (Compared to Sham)

TABLE 8 Group XSSI YSSI PSSI Control  3.43 ± 0.76^(#) 4.51 ± 1.31 7.31 ±2.03 Sham 5.61 ± 1.81 5.64 ± 1.01 9.75 ± 1.23 T-100 2.83 ± 0.41 3.21 ±1.74 5.35 ± 2.65 ^(#)p < 0.05 (Compared to Sham)

The length of periosteum and endosteum as shown in Table 6, the bonemass, density and thickness of cortical bone as shown in Table 7, andthe index of bone strength as shown in Table 8 were not significantlydifferent among control and T-100 groups.

According to the examples above, the mycelium of C. Sinensis couldsuppress the formation of osteoclasts in vitro, thereby might inhibitbone resorption (bone erosion) in vivo. In the QCT experiment, since themycelium of C. Sinensis could slow down the decrease of spongy bonedensity, thus the mycelium of C. Sinensis was able to suppressosteoporosis. In addition, measuring DEXA, pQCT had found that the bonedensity was significantly different. Given from the in vitro and in vivostudies above, the mycelium of C. Sinensis could suppress osteoporosis.

The effect of the present invention.

As discussed above, drug containing the mycelium of C. Sinensis,especially cultured C. Sinensis, of the present invention, could inhibitthe formation of osteoclasts, slowed the decrease of bone density andthus inhibited osteoporosis when administered.

1. A method for treating osteoporosis comprising administering to apatient in need of treatment, an effective amount of a compositioncomprising a water extract of an organic solvent extract of culture ofCordyceps sinensis as an effective ingredient.
 2. The method accordingto claim 1, wherein the treatment of osteoporosis is made by suppressinga decrease of spongy bone density.
 3. The method according to claim 1,wherein the Cordyceps sinensis is mycelium of Cordyceps sinensis.
 4. Themethod according to claim 1, wherein at least one organic solvent isdiethyl ether.
 5. The method according to claim 1, wherein at least oneorganic solvent is selected from the group consisting of alcohol, ester,alkane and halogenated alkane.
 6. The method according to claim 5,wherein the alcohol is methanol or ethanol.
 7. The method according toclaim 1, wherein said administering comprises at least oneadministration route selected from the group consisting of intravenous,oral, intra-tissue, eye, nose, and colon.
 8. The method according toclaim 1, wherein said composition is in a form selected from the groupconsisting of powder, tablet, capsule, granule, droplet, solution,syrup, and sublingual tablets.